Random mutagenesis identifies novel genes involved in the secretion of antimicrobial, cell wall-lytic enzymes by Lactococcus lactis.
Lactococcus lactis is a gram-positive bacterium that is widely used in the food industry and is therefore desirable as a candidate for the production and secretion of recombinant proteins. Previously, we generated a L. lactis strain that expressed and secreted the antimicrobial cell wall-lytic enzyme lysostaphin. To identify lactococcal gene products that affect the production of lysostaphin, we isolated and characterized mutants generated by random transposon mutagenesis that had altered lysostaphin activity. Out of 35,000 mutants screened, only one with no lysostaphin activity was identified, and it was found to contain an insertion in the lysostaphin expression cassette. Ten mutants with higher lysostaphin activity contained insertions in only four different genes, which encode an uncharacterized putative transmembrane protein (llmg_0609) (three mutants), an enzyme catalyzing the first step in peptidoglycan biosynthesis (murA2) (five mutants), a putative regulator of peptidoglycan modification (trmA) (one mutant), and an uncharacterized enzyme possibly involved in ubiquinone biosynthesis (llmg_2148) (one mutant). These mutants were found to secrete larger amounts of lysostaphin than the control strain (MG1363[lss]), and the greatest increase in secretion was 9.8- to 16.1-fold, for the llmg_0609 mutants. The lysostaphin-oversecreting llmg_0609, murA2, and trmA mutants were also found to secrete larger amounts of another cell wall-lytic enzyme (the Listeria monocytogenes bacteriophage endolysin Ply511) than the control strain, indicating that the phenotype is not limited to lysostaphin.
Tan
Yu Pei
YP
Infectious Diseases Program, Cells and Tissue Domain, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia.
Giffard
Philip M
PM
Barry
Daniel G
DG
Huston
Wilhelmina M
WM
Turner
Mark S
MS
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002876:Chromosomes, Bacterial; D004251:DNA Transposable Elements; D023061:Gene Order; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D008248:Lysostaphin / Q000378:metabolism*; D016254:Mutagenesis, Insertional
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D008248:Lysostaphin